PROJECT SUMMARY/ABSTRACT Multiple cases of MEHMO syndrome, an X-linked intellectual disability syndrome caused by mutations in EIF2S3 that encodes a core member of the cellular protein synthesis machinery, have recently been identified; however, the causal links between misregulated protein synthesis and human disease remain poorly understood. EIF2S3 encodes the ? subunit of translation initiation factor 2 (eIF2?) that, along with the eIF2 ? and ? subunits, GTP, and the initiator Met-tRNAiMet, plays a critical role in selection of the translation start site, indicating that MEHMO patients likely incur changes in the level of protein synthesis on both a global and gene specific level. The acronym MEHMO denotes the symptoms of this syndrome: Mental deficiency, Epilepsy, Hypogenitalism, Microcephaly, and Obesity. It remains unknown how changes in gene expression incurred through mutations in eIF2? result in the presentation of MEHMO syndrome. Thus, there is a critical need to determine the underlying molecular mechanism governing the phenotype and symptoms of MEHMO patients. The long-term goal of these studies is to contribute to the identification of key factors that serve to promote MEHMO syndrome, so that improved strategies can be developed for the treatment of MEHMO patients. The overall objective in this application is to determine how misregulation of protein synthesis contributes to the symptoms of MEHMO syndrome. In Aim 1 CRISPR Cas9 genome editing will be used to generate isogenic control iPSCs expressing wild-type eIF2? from iPSCs that were derived from a MEHMO patient and express mutant eIF2?. Generation of isogeneic control iPSCs from patient iPSCs will enable investigations in the patient's genetic background to determine how mutation of eIF2? differentially modulates gene expression. In Aim 2 ribosome profiling, an informatics-based, genome-wide approach, and additional validation techniques will be used to identify mRNAs subject to changes in start codon selection and translational efficiency that may contribute to the symptoms of MEHMO syndrome. In Aim 3 wild-type and eIF2? mutant iPSCs will be differentiated into neuronal cells that will be utilized in multidisciplinary analyses, including ribosome profiling and electrophysiological readings, geared toward understanding the changes in neuronal cell homeostasis and function incurred through eIF2? mutation. These studies will provide a richer understanding of how protein synthesis is modulated upon mutation of eIF2? and the role that translational control of gene expression plays in the manifestation of MEHMO syndrome.